The use of nucleic acids has proved effective for altering the state of a cell. The introduction of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) into a cell can be used to up- or down-regulate the expression of particular genes in the cell, thereby, impacting one or more biochemical pathways. Of the nucleic acid-based technologies used to alter cell physiology, RNA interference (RNAi) is the general term given for regulating the expression of genes at the post-transcriptional level in diversified organisms. RNAi gene silencing can be accomplished using homologous short (21-23 bp) dsRNA fragments known as short interfering or “siRNA.” When a long dsRNA is introduced into a cell line, the cellular enzyme Dicer will cleave it into short interfering RNA (siRNA) molecules. This short interfering RNA molecule is now called the guided RNA. The guided RNA will guide the RNA-Induced-Silencing-Complex (RISC) to the homologous target mRNA. Once it forms a hybrid structure to the homologous mRNA sequence, the RISC will cleave the mRNA. As a result, protein that is encoded by the mRNA will no longer be produced, thereby causing the silencing of the gene. RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
However, a major obstacle for the development of a RNAi-based therapeutic approaches for brain pathologies is the blood-brain barrier (BBB). The brain is shielded against potentially toxic substances by the presence of two barrier systems: the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). The BBB is considered to be the major route for the uptake of serum ligands since its surface area is approximately 5000-fold greater than that of BCSFB. The brain endothelium, which constitutes the BBB, represents the major obstacle for the use of potential drugs against many disorders of the CNS. As a general rule, only small lipophilic molecules may pass across the BBB, i.e., from circulating systemic blood to brain. Many drugs that have a larger size or higher hydrophobicity show promising results in animal studies for treating CNS disorders.
Besides direct intrabrain administration, different strategies have been described for achieving gene silencing in the CNS by means of systemically-administered RNA interfering molecules. For instance, Kumar et al. (Nature, 2007, 448:39-44) have described conjugates of siRNA and a peptide derived from the rabies virus glycoprotein comprising a nonamer arginine and their ability to silence gene expression in the brain after intravenous injection. Xia et al. (Pharmaceutical Research, 2007, 24:2309-2316) have described conjugates comprising a biotinylated siRNA and a conjugate comprising avidin-anti-transferrin receptor antibody which are capable of silencing gene expression in the central nervous system after systemic delivery. WO200979790 describe conjugates comprising siRNA and a series of peptides collectively known as Angiopeps which are capable of crossing the blood-brain barrier by receptor-mediated transcytosis using the low-density lipoprotein receptor-related protein-1 (LRP-1) and which allows the delivery to the CNS of systemically administered conjugates comprising said peptides. WO2007107789 describes the use of compounds capable of causing RNA interference and which are specific for targets present in the CNS and the delivery to the CNS by the use of intranasal administration.
However, while all these systems allow the delivery of systemically administered siRNAs to the CNS, they do not allow delivery to specific cell types within the brain. In fact, no delivery system has been described to date which allows delivery of a therapeutic agent to a specific cell type within the CNS. The possibility of delivering siRNAs of known specificity to the central nervous system will be usesful for the treatment of diseases which are caused by an undesired activity/expression of a given gene, including depression, cognitive disorders, Parkinson's disease, Alzheimer's disease, etc.
Depression is recognized as a disease of the central nervous system. Depression is both biologically and genetically a heterogeneous disorder, with symptoms manifested at the psychological, behavioural and physiological level. Moreover, depression shows a high degree of co-morbidity with anxiety disorders and anxiety itself (typically anticipatory anxiety) is one of the most prevalent symptoms in depressive patients. Indeed, most anxiety disorders are also treated with antidepressant drugs.
The first drugs used in the treatment of major depression were the tricyclic antidepressants (TCAs) of the imipramine type and the monoamine oxidase inhibitors (MAOIs). These drugs were discovered in the late 1950s and proved efficacious, yet they presented a number of severe side effects that led to the development of new drugs, such as the Selective Serotonin Reuptake Inhibitors (SSRIs) or the selective Serotonin and Noradrenaline Reuptake Inhibitors (SNRIs).
The discovery that TCAs (and later, SSRIs and SNRIs) inhibited the reuptake of the monoamines serotonin (5-HT) and noradrenaline (NA) into the presynaptic cell, increasing levels of 5-HT within the synaptic cleft, thereby enhancing their activity at postsynaptic receptor, led to the first hypotheses of the ethiology of depression, i.e., that it was caused by a deficit of the activity of these monoaminergic neurotransmitter systems in the brain. Ever since, all marketed antidepressant drugs have targeted serotonergic and/or noradrenergic transporters or receptors.
5-HT receptors are located on the cell membrane of nerve cells and other cell types in animals. With the exception of the 5-HT3 receptor, all other 5-HT receptors are G protein coupled seven transmembrane (or heptahelical) receptors that activate an intracellular second messenger cascade. Some of the identified 5-HT receptors include the 5-HT1A and the 5-HT1B/1D receptors expressed, presynaptically on serotonin neurons (autoreceptors) and on neurons postsynaptically located to 5-HT nerve terminals. The 5-HT receptor more directly linked with the antidepressant effects of SSRIs has been the 5-HT1A receptor.
New antidepressant drugs are now being registered with mechanisms of action based on relatively selective norepinephrine reuptake inhibition (NARI), e.g. reboxetine, or in the dual blockade (SNRIs), such as venlafaxine or duloxetine. Other drugs, such as nefazodone, trazodone or mirtazapine have a weaker action at monoamine transporters and block monoaminergic receptors instead.
However, notwithstanding the commercial success of SSRIs, these compounds have two major limitations: 1) only 60% of patients experience a therapeutic response (reduction to half of baseline severity), and 2) response occurs only after several weeks of continued treatment. This is due to a negative feedback mechanism that takes place in the pre-synaptic neuron. Briefly, high serotonin levels induced by the blockage of serotonin reuptake will not only activate the post-synaptic serotonin receptors, but also activate presynaptic autoreceptors, which serve as a feedback sensor for the cell. The activation of 5-HT1A autoreceptor by 5-HT (also called pre-synaptic 5-HT1A receptor or pre-synaptic 5-HT1AR), or selective agonists, suppresses cell firing and impulse-dependent 5-HT release, whereas 5-HT1B receptors control 5-HT synthesis and release at terminal level. Both, 5-HT1A and 5-HT1B receptors, are also localized on neurons postsynaptic to 5-HT nerve terminals, mainly in cortico-limbic areas. The increase of extracellular 5-HT produced by reuptake blockade of sertraline (SERT, a SSRI) activates pre-synaptic 5-HT1A receptor in the midbrain raphe nuclei, suppressing cell firing and terminal release, an effect that attenuates the extracellular 5-HT increase produced by reuptake blockade. 5-HT1B autoreceptors exert a similar negative feedback at a local level. Following repeated administration of SSRIs, 5-HT1A autoreceptors desensitize, which enables serotoninergic neurons to recover cell firing and leads to an increase in extracellular 5-HT, to a level higher than that seen after single treatment. These (slowly proceeding) neurophysiological adaptations of the brain tissue are not only the reason why usually several weeks of continuous SSRI use are necessary for the antidepressant effect to become fully manifested, but also why increased anxiety is a common side effect in the first few days or weeks of use. It is known that the blockade of these negative feedback mechanisms with 5-HT1A and/or 5-HT1B receptor antagonists potentiates the 5-HT increase produced by SSRIs and, therefore, might serve to accelerate the clinical effects of SSRIs.
The pharmacological strategy to accelerate the antidepressant response by blocking the action of pre-synaptic 5-HT1A receptors during SSRI administration was tested using (±)pindolol. This compound is a β1-2 adrenergic receptor antagonist with a putative antagonistic action on 5-HT1A receptors. (±)Pindolol antagonized several actions mediated by the activation of central 5-HT1A receptors, such as hypothermia or hormonal secretion. In general, the addition of pindolol to SSRIs accelerates the antidepressant response. However, although pindolol has been shown in some studies to partially occupy 5-HT1A receptors in the human brain at clinical doses, other studies have found a low occupancy. Additionally, it is not to be forgotten that 5-HT1A receptors are localized on the serotoninergic neurons as well as on neurons postsynaptic to the serotoninergic neurons. Indeed, an important concern is the lack of selectivity of these agents for pre-synaptic versus postsynaptic 5-HT1A receptors: the full blockade of postsynaptic receptors may cancel the increased transmission through forebrain 5-HT1A receptors produced by antidepressant drugs.
Thus, despite the advances made in the development of antidepressants, there is still the need of alternative compounds which specifically act on the pre-synaptic 5-HT1A receptors.
Parkinson's disease (PD) is a degenerative disorder of the central nervous system that often impairs the patient's motor skills, speech, and other functions (Olanow). The symptoms of Parkinson's disease result from the greatly reduced activity of dopaminergic cells in the pars compacta region of the substantia nigra (SNpc) (Olanow, Dawson). These neurons project to the striatum and their loss leads to alterations in the activity of the neural circuits within the basal ganglia that regulate movement, in essence an inhibition of the direct pathway and excitation of the indirect pathway. The direct pathway facilitates movement and the indirect pathway inhibits movement, thus the loss of these cells leads to a hypokinetic movement disorder. The lack of dopamine results in increased inhibition of the ventral anterior nucleus of the thalamus, which sends excitatory projections to the motor cortex, thus leading to hypokinesia.
PD is characterized by a progressive loss of dopaminergic neurons in the SNpc and the presence of intracellular inclusions designated as Lewy bodies (LB). Neurochemically, PD is marked by mitochondrial complex I dysfunction and increased indices of oxidative stress. Several pathogenic mechanisms have been proposed for PD including oxidative and nitrosative stress, mitochondrial dysfunction, protein misfolding and aggregation, and apoptosis. PD is mostly sporadic but some of the PD cases have been shown to be familial-linked. The first familial-linked PD gene identified was α-synuclein (α-syn) which in fact is the major component of LB in all PD patients. The normal function of α-synuclein is poorly understood. α-Synuclein can bind to lipids and, in neurons, is associated with presynaptic vesicles and the plasma membrane, possibly via lipid rafts. The deposited, pathological forms of α-synuclein are aggregated and show lower solubility than the normal protein. Three point mutations have been described to cause familial PD, but also duplications and triplications of the SNCA gene have been reported to be responsible of PD and Lewy body disease. Therefore, even without sequence variants, α-synuclein dosage can be causal for Lewy body disease.
α-Synuclein affects mitochondria and probably induces apoptosis. In fact, there is accumulating evidence for a close relationship between α-synuclein and oxidative damage: overexpression of mutant α-synuclein sensitizes neurons to oxidative stress and damage by dopamine and complex I inhibitors, resulting in increased protein carbonylation and lipid peroxidation in vitro and in vivo. Conversely, dysfunction of mitochondrial complex I has been associated to sporadic forms of PD. Complex I dependent oxidative damage and defective mitochondrial function is a main cause of neuronal degeneration and cell death in PD. Thus impaired mitochondrial function and ROS production increases the cytochrome c pool level in the mitochondrial intermembrane space, allowing its rapid release when the cell death agonist Bax is activated.
To sum up, the scenario in PD would be a situation of neuronal mitochondrial dysfunction with increase ROS production that on one hand would increase α-synuclein accumulation and on the other would activate Bax-mediated cell death. Further, α-synuclein accumulation, in turn, would increase cellular ROS production and induction of neuronal degeneration.
The most widely used treatment for PD is L-DOPA in various forms. However, only 1-5% of L-DOPA enters the dopaminergic neurons. The remaining L-DOPA is often metabolised to dopamine elsewhere, causing a wide variety of side effects. Dopa decarboxylase inhibitors like carbidopa and benserazide are also used for the treatment of PD since they help to prevent the metabolism of L-DOPA before it reaches the dopaminergic neurons and are generally given as combination preparations of carbidopa/levodopa and benserazide/levodopa. Moreover, dopamine agonists are moderately effective and act by stimulating some of the dopamine receptors. However, they cause the dopamine receptors to become progressively less sensitive, thereby eventually increasing the symptoms.
Antisense approaches might also be helpful, and have been reported to work in the rat and mouse brain. This approach is predicated on the idea that α-synuclein really is dispensable for CNS function in humans, as it appears to be in the mouse but perhaps even a modest decrease in protein levels would be enough to decrease PD progression.
However, despite the advances made in the development of PD therapeutics, there is still the need of alternative compounds which specifically are capable of preventing the reduced activity of dopaminergic cells in the pars compacta region of the substantia nigra.
Mesocortical and mesolimbic dopamine (DA) systems play a crucial role in many psychiatric disorders including schizophrenia. A general enhancement of brain dopaminergic neurotransmission in schizophrenia was suggested by pharmacologic evidence (Seeman and Lee, 1975; Creese et al, 1976). Current views, however, indicate a hyperactivity of subcortical DA transmission together with a hypoactive mesocortical. The overall efficacy of classical (DA D2 receptor antagonists) and atypical antipsychotics (APDs, preferential 5-HT2A/2C vs. DA D2 receptor antagonists) to treat positive (psychotic) symptoms is similar. In contrast, some agents of the latter group, and particularly clozapine, are superior to classical antipsychotics for the treatment of negative symptoms and cognitive impairment. This clinical feature has been related, at least in part, to the ability to increase DA release in the mesocortical pathway, an effect induced by atypical—but not classical—antipsychotics. Indeed, an optimal prefrontal DA function is crucial for working memory and executive functions.
DA release in mesocortical and mesolimbic DA pathways is regulated by several factors. Firstly, it depends on the firing mode (tonic/phasic) of VTA DA neurons. Secondly, it is tightly regulated by the activation of somatodendritic and terminal D2/3 autoreceptors which control cell firing and DA release. Finally, the DA transporter (DAT)-mediated reuptake is one of the key mechanisms that define decay kinetics of extracellular DA concentrations. Previous studies indicate a different density of DAT in PFC and striatum.
Moreover, noradrenaline (NA) axons may contribute to the removal of DA from the extracellular brain space, since the NA transporter (NAT) shows a similar affinity for NA and DA. Thus, NAT inhibitors preferentially increase the extracellular DA concentration in the medial PFC (mPFC) compared to caudate and nucleus accumbens (NAc). Hence, NA axons from locus coeruleus (LC) neurons may contribute to regulate the extracellular DA concentration in PFC either by taking up or co-releasing DA. Some researchers have shown the effects of a new combination treatment based on NA-targeting drugs (NAT inhibitor plus α2-adrenergic antagonist) to selectively enhance mesocortical DA transmission.
However, there is still a need for compounds capable of enhancing mesocortical DA transmission.